1. Field of the Invention
This invention relates to a continuous and relatively instantaneous antibody-based method for detecting a target moiety and a flow immunosensor apparatus capable of conducting the method.
2. Description of the Prior Art
Today, for reasons of health and safety, it is necessary to be able to rapidly detect the presence of moieties in an environment or samples which are themselves dangerous or are associated with materials which can be dangerous. These moieties may be in a medicine, a food or a sample of body fluid. In addition, these moieties can be in the air or water. The moieties are referred to herein as a target or target moiety.
The target may be something dangerous to health such as a toxic material present in a food, or a toxic agent present in the air or in the water supply. In addition, the target can be a chemical indicating the presence of something dangerous to human welfare such as vapors exuded by explosives and illicit drugs. Further, the target can be a specific component or metabolite found in animal body fluids. These later targets can indicate presence of disease or simply a physical condition.
Many tests are available or proposed for detecting target moieties under laboratory conditions. Toxins in food or water are detected by chemical tests conducted on a batch basis. In addition, toxins in air or water can be detected by continuous processes but, to date, these processes need a relatively large target sample to accomplish the purpose. In addition, the tests do not distinguish between or are blind to many targets. Many batch tests exist for the detection and identification of metabolites or components of body fluids. Most of these tests do not provide substantially instantaneous answers or rapid while-you-wait answers.
Real-time tests are defined here as tests which will provide an answer within two or three minutes of a sample being taken and introduced to the test. Tests which require removing a sample from an environment and subjecting it to multiple handling and transfer operations with developing or incubation time for a reading are not considered real-time tests. As an illustrative example only, removal of a sample from a flowing air or water stream and providing an answer in less than a minute so that the conditions indicated by the test are indicative of the conditions in the environment is considered a real-time test.
Two areas of high interest today are the detection of drugs and the detection of explosive materials. Although the chemical problems for detecting these two types of materials are markedly different, the physical problems are often the same. Both require real-time tests. Similarly, there is also a need for a real-time method of detecting the presence of a moiety in a sample-by-sample situation so a sample can be quickly characterized and appropriate action taken. This might be a simple qualitative test at a crime scene to identify a sample as a drug, a sample as blood, or a sample as semen.
As acts of terrorism, bombs have been placed aboard aircraft or in buildings. Explosive devices are usually hidden in small, enclosed and restricted spaces. Defense against such acts requires detection of the explosive materials in the hiding place. Similarly, illegal drugs are smuggled into a country, building or other secured area by being hidden in small, enclosed and restricted spaces. The vapor pressure of most drugs and explosives is such that very small amounts are available for detection by sampling air. Detection of such small amounts of target vapors of specific targets requires discriminating and sensitive equipment, test methods and materials.
U.S. Pat. No. 4,818,870 to Griffith et al. describes an air sampling probe used with "sniffer" devices. These devices attempt to detect vapors from drugs and explosives. The detector in Griffith's invention is a mass analyzer. U.S. Pat. No. 4,866,439 discloses a means for detecting explosives using an electron capture detector. U.S. Pat. Nos. 4,849,628; 4,776,409 and 4,884,839 describe atmospheric samplers which use ionization sources or photoionization detectors. In U.S. Pat. No. 4,360,776, Bauman describes the use of antibodies in an electron spin resonance technique to detect target agents.
Monoclonal antibody technology has markedly changed modern chemical and medical analytical techniques. Since monoclonal antibody technology has made highly specific antibodies available in large quantities, columns containing supports coated with antibodies have come into general use for the purification of antigens.
Antibody-based detection systems such as ELISAs (Enzyme Linked ImmunoSorbent Assays) and radioimmunoassays have been developed to take advantage of antibody specificity and sensitivity. However, while sensitive, these techniques are time-consuming and usually require multiple manipulations and/or reagent additions. Antibodies, due to their inherent sensitivity and selectivity, are natural candidates for use as the detection element in biosensors.
Biosensors incorporating antibodies have been classified as immunosensors. Most of the immunosensors reported to date (Ives et al., Applied Biotechnology Laboratory, Mar. 10, 1989; Andrade et al., U.S. Pat. No. 4,368,047 (1983); Place et al., Biosensors, 1, 321 (1985); Tromberg et al., Anal Chem., 59, 1226 (1987); Thompson, R. B. and F. S. Ligler. NRL Memorandum Report 6182, 1988) rely on the association of antibody with antigen and are configured as direct binding or sandwich assays for detection of large molecules or as competition assays for the detection of small molecules.
The widespread use of ELISAs and affinity chromatography have stimulated theoretical consideration of the kinetics of antigen-antibody binding at the solid-liquid interface (Lew, J. ImmunoL Methods, 72, 171 (1984); Lundstrom et al., J. Theor. Biol., 110, 195 (1984); Nygren et al., J. Immunol Methods, 101, 63 (1987); Stenberg et al., J. Immunol. Methods, 113, 3 (1988)). The rate of association for antigen and antibody is determined to be a function of the density of the immobilized component, antibody affinity, geometry of the substrate, and concentration of the free component. In the studies cited above, the antigen is bound to the surface and the antibody is free in solution.
For multivalent systems, Stenberg et al. (1988), cited above, have shown that the association rate of antibodies and antigens at the surface of macroscopic particles is limited by the diffusional mass transfer as long as the surface reaction is fast compared to the diffusion rate. In the diffusion limited region, the rate is inversely proportional to the sphere radius.
Ives et al. (1989), cited above, briefly discuss the displacement of a labelled antigen by an excess of unlabeled antigen under the assumption that it is an equilibrium exchange reaction. As long as the antigen is monovalent and of low molecular weight, Ives et al. concludes that the exchange will require 15-20 minutes.
For an antigen-antibody interaction, the dissociation rate is normally considered to determine the strength of binding. For dissociation of IgG and Fab' antibodies from immobilized antigens, rate constants in the range of 10.sup.-4 -10.sup.-5 sec.sup.-1 have been obtained (Liu et al., IEEE Trans. of Biomedical Eng., Vol. BME-33, No. 2, February 1986; Nygren, 1987, cited above; Mason et al., Biochem J., 187, 1 (1980); Werthen et al., J. ImmunoL Methods, 115, 71 (1988)). For the time intervals typical of immunoassay, the binding can thus be considered as irreversible.
In the studies of Nygren (1987), cited above, over a course of several hours, the only dissociation seen of antibody bound to immobilized antigen was in the presence of excess antigen. The displacement of bound antigen from antibody affinity columns upon the addition of excess free antigen is also well known.
S Aizawa et al., Transducers '87, 783 (1988), report "remarkable" displacement of peroxidase-labelled IgG antibody from immobilized antigen using micromolar quantities of free antigen. Aizawa et al.'s report was incomplete because neither the time nor the ratio of antibody to free antigen was reported.
The hit-and-run immunoassay (Warden et al., Anal. Biochem., 162, 363 (1987) is an immunoassay also conducted on a column support. A problem with this hit-and-run assay is that it is not a continuous flow system. When antigen is introduced to the column, it is allowed to remain there for 5 minutes. Thus in a 5-minute period, only one column volume of sample can be tested. Also, during this time, diffusion rather than flow rate determines the number of antigen-antibody encounters. In addition, antigen is immobilized on the column and a fluorescent marked Fab' fragment of IgG is bound to it This Fab' fragment is claimed to be in an equilibrium-binding condition so that it is continually bound to individual immobilized antigens, released, and rebound. When sample containing antigen is introduced, the released Fab' fragment binds to it over the 5-minute period and the soluble fluorescent complex flows downstream to the fluorescence detector.
Antibodies have been applied to the detection of illicit drugs and explosives. In U.S. Pat. No. 4,353,886, Lukens et al. describe a field test for narcotic vapors. The test uses antibodies mounted on a test plate to detect the vapor. The test is a batch type system requiring the exposure and development of different plates. The system can not continually monitor an environment.
Many techniques have been developed for detecting drugs either singularly or in groups. In U.S. Pat. No. 4,235,864, Kaul et al. describe a method of detecting and identifying multiple drugs found together. A radiolabelled conjugate of ecgonine is used to detect cocaine in a traditional radioimmunoassay. Methods for the preparation of the antibodies for many of the illicit drugs are well known and some are available from commercial sources.
The existing techniques using antibodies do not have the capability to continually monitor an environment or provide a response in real time. Existing systems are semi-batch processes depending on direct binding, competitive binding, or equilibrium exchange reactions.